The major goals of this project are 1) to create a genome-based description of human erythropoiesis, and 2) to use that description to develop novel therapies for diseases involving erythroid cells. Erythroid diseases encompass a broad range of anemias, hemoglobinopathies and malarial syndromes. Accomplishments achieved during the last year: 1. Hembase: a description of the human erythroid transcriptome. Previously, we reported the sequencing of human erythroblast libraries to generate an informatic database describing the erythroid transcriptome. That database, called Hembase (http://hembase.niddk.nih.gov/), is comprised of homology comparisons from our Expressed Sequence Tag (EST) collection with sequences contained within other publicly available databases. During the last year, the Hembase reformatting began in 2003 was completed. The new Hembase was launched on the Internet in late 2003 as the first human genome portal placed in the public domain. The current version of Hembase now consists of 16,000 searchable files with direct links to the primary sequence data as well as specific links to three major human genome browsers currently in the public domain. Conceptually, a genome portal format was created to provide search-current information to the users rather than static information that may no longer be current at the time of each query. For clinical investigators, the database was further refined to include categories of ?red cell topics? describing genetic subsets involved in specific erythroid pathologies (e.g. hemolysis related enzymes). 2. Studies of signaled activation of fetal hemoglobin among primary human erythroblasts. Information gathered in Hembase (above) led us to hypothesize that the pattern of hemoglobin accumulation during adult erythropoiesis is directly related to the proliferation of the cells. We further postulated that regulation of signal transduction cascades and growth among fully committed erythroid cells may be used to increase levels of fetal hemoglobin. In this context, we developed a standard experimental assay of cultured human erythroid progenitor cells obtained from normal volunteers to identify cytokines capable of modulating erythroid growth and fetal hemoglobin production during adult erythropoiesis. Stem cell factor (SCF) was identified as having significant effects upon erythroid growth and fetal hemoglobin production even among committed proerythroblasts. Conceptually, these results suggest that new therapies aimed at the manipulation of erythroblast signaling cascades may be developed for the treatment of sickle-cell diseases and beta-thalassemias. During the last year, we continued studies of the SCF effects upon fetal hemoglobin modulation. Quantitative polymerase chain reaction demonstrated a transcriptional mechanism for the hemoglobin changes (increased gamma-globin mRNA). The primary eryhroblasts were additionally cultured in the presence of several growth-related signaling pathway inhibitors to determine which pathways are involved in the SCF effects. The MEK1/2 inhibitor (PD98059) inhibited the SCF effects as determined by a linear dose response in levels of fetal hemoglobin. Western blot analyses confirmed that SCF led to phosphorylation of MEK and p44MAPK in proerythroblasts. These results correlate with classic studies that associated increased fetal hemoglobin and malignant erythropoiesis, and further support the pursuit of erythroblast signal transduction as a treatment modality for beta hemoglobinopathies. 3. Continued characterization of novel erythroid transcription. Studies aimed toward a more complete understanding of growth-inhibition during terminal cellular differentiation remain an active area of translational research aimed toward the treatment of leukemias and other growth-related diseases. Ex vivo cultures of erythroid cells provide one of the only experimental system for the study of terminal cellular differentiation using primary cells from human donors. Relatively little in known about the mechanism(s) that inhibit growth during terminal erythroid differentiation. We previously determined that erythroid gene patterning is useful for the identification of those elements that are dysregulated in patients with myelodysplastic syndromes. We have more recently begun to screen the erythroid transcriptome for growth inhibiting genes that are normally expressed during terminal erythroid differentiation. The NIX gene was characterized in this context (see 2003 annual report). During the last year, we reported the cloning and characterization of a novel growth-related gene (EEG-1) that is located on the short arm of chromosome 12. EEG-1 expression was highly regulated with increased expression at the stage of differentiation associated with the onset of global nuclear condensation and reduced cell proliferation. Transfection of EEG-1 was associated with significantly increased levels of apoptosis, and the transfected protein co-localized with aggregated mitochondria. Stable transfection assays additionally demonstrated decreased growth in those cells expressing EEG-1 at higher levels. We proposed that the regulated expression of EEG-1 is involved in the orchestrated regulation of growth that occurs as erythroblasts shift from a highly proliferative state toward their terminal phase of differentiation. We continue to believe that gene discovery projects (e.g. NIX and EEG-1) should significantly enhance efforts aimed toward understanding the regulation of growth during terminal cellular differentiation among mammalian cells. 4. Based upon our interests in erythroblast signal transduction as a modulator of hemoglobin production, we initialed studies aimed toward globin gene regulation. Using Hembase, we identified and an alternate delta-globin mRNA (Alt-d). Alt-d mRNA shares the same coding region, splicing pattern, downstream untranslated region, and site of polyadenylation with the previously defined delta-globin (Delta) mRNA. Alt-d mRNA encodes an additional 145 nt in the upstream untranslated region, suggesting an alternative site of transcriptional initiation and transcription through the previously defined promoter, which contains several protein-binding motifs and a TATA box. Northern blot and PCR analyses demonstrated a restricted expression of Alt-d in erythroid cells. This discovery of delta globin promoter transcription suggests that the genome-based approach is useful for identifying novel globin gene regulatory mechanisms. Through collaboration, the experimental model was also used to provide a human correlate of findings involving GATA-1 gene regulation originally identified in the murine system.